The invention relates to an improved and simplified process for purifying organophosphorus hydrolase enzyme (xe2x80x9cOPHxe2x80x9d) from a recombinant host cell, that expresses this enzyme.
The threat of exposure to chemical warfare (xe2x80x9cCWxe2x80x9d) agents is of increasing concern for both the armed forces and for civilian populations that might be potentially targeted by terrorists. For this reason, there is an acute need to develop and improve technology for decontamination of CW agents. This is especially true for the class of CW agent known as nerve agents or nerve gases. One class of nerve agents with a high level of potential lethality is the class that includes organophosphorus-based (xe2x80x9cOPxe2x80x9d) compounds, such as Sarin, Soman, and the VX. Such CW nerve agents can be absorbed through inhalation and/or through the skin by the victim. The OP nerve agents typically manifest their lethal effects against animals and people by inhibiting acetylcholine esterase (xe2x80x9cAChExe2x80x9d) enzyme at neuromuscular junctions between nerve endings and muscle tissue. An excessive buildup of the neurotransmitter, acetylcholine, can result in paralysis and death in a short time.
In addition to the concerns about CW agents, there is also a growing need in industry for decontamination of industrial OP-based insecticides, for example, acetylcholinesterase-inhibiting pesticides such as parathion, paraoxon and malathion, among others. Thus, it is very important to be able to effectively detoxify a broad spectrum of toxic OP compounds on contaminated surfaces and sensitive equipment.
Currently, the U.S. Army uses a nerve agent decontamination solution, DS2, which is composed (by weight) of 2% NaOH, 28% ethylene glycol monomethyl ether, and 70% diethylenetriamine (Richardson, G. A. xe2x80x9cDevelopment of a package decontamination systemxe2x80x9d, EACR-1 310-17, U.S. Army Edgewood Arsenal Contract Report (1972), herein incorporated by reference). Although this decontamination solution is effective against OP nerve agents, it is quite toxic, flammable, highly corrosive, and releases toxic by products into the environment. Thus, there is a need for an alternative decontamination technology that is both effective and non-hazardous to personnel, sensitive equipment, and/or the environment.
One potential alternative to DS2 is enzyme-mediated decontamination. Enzymes are biocatalysts that are typically non-toxic, biodegradable, non-corrosive and can be economically produced in the desired quantities. Suitable enzymes are those that effectively catalyze the degradation of OP-based toxic compounds, including CW agents. For example, a class of enzymes known as organophosphorus anhydrolases (xe2x80x9cOPAAxe2x80x9d)(EC 3.1.8.2) can broadly catalyze the hydrolysis of a variety of OP compounds, including fluorinated xe2x80x9cG-typexe2x80x9d nerve agents. (See, e.g., Landis, W. G., et al., 1987, J. Appl. Toxicol. 7:35-41; DeFrank, J. J., et al., 1993, Chem. Biol. Interact., 87: 141-148; both incorporated by reference herein in their entireties). This enzyme, however, does not detoxify V-type CW nerve agents.
Another potentially useful OP-degrading enzymes is organophosphorus hydrolase (OPH; EC 3.1.8.1). OPH is particularly desirable because it is the only well-characterized enzyme that can hydrolyze both United States and Russian Federation types of VX nerve agents. Two common sources for OPH enzyme, to date, are the identical opd genes isolated from Pseudomonas diminuta MG and the Flavobacterium sp. strain ATCC 27551. The P. diminuta MG opd gene was isolated by McDaniel et al., 1989, J. Bacteriol., 170:2306-2311, incorporated by reference herein in its entirety. The McDaniel et al. opd gene is referenced in Genebank, with ascension number M20392, and incorporated by reference herein in its entirety, as follows.
LOCUS PSEPTE 1322 bp DNA BCT Apr. 21, 1996
DEFINITION Plasmid pCMS1 (from P. diminuta) phosphodiesterase (opd) gene, complete cds.
ACCESSION M20392
NID g151517
VERSION M20392.1 GI:151517
The open reading frame of the opd gene, as reported by McDaniel et al., contains 975 bases which encode OPH polypeptide of 325 amino acid residues with a molecular mass of 35 kDa. Mulbry, W. et al., 1989, J. Bacteriol., 171, 6740-6746, incorporated by reference herein in its entirety, also cloned the opd gene, but that clone lacked 4 amino-terminal residues (ser-ile-gly-thr or SIGT), relative to the opd gene described above.
Previously developed procedures for purification of OPH have broadly required four purification steps. See, for example, Omburo, et al., 1992 J. Biol. Chem. 267:13278-13283. Omburo, et al., described two precipitation steps and two chromatography steps as listed below:
1. protamine sulfate (0.4%) precipitation and clarification;
2. ammonium sulfate (45%) precipitation with resuspension and dialysis;
3. Ultragel ACA 54(trademark) or Sephacryl S-200(trademark) gel filtration;
4. DEAE-Sephadex A-25(trademark) or DEAE-Sephacel(trademark) ion-exchange chromatography.
Another problem is that the OPH enzyme has a limited shelf-life in aqueous solution, and the previously employed process exposes the OPH enzyme to an aqueous environment for a prolonged time period. For example, at room temperature, the OPH enzyme loses more than 50% of its catalytic performance within 4-5 hours, and the above-described 4-step process requires 4-5 days for completion. Given the number of steps and the prolonged processing times heretofore required, it is clear that this previously employed process has failed to fulfil the need for economical production of OPH enzyme at high yield. In particular, the above-described 4-step process only produces from 1-5 mg of OPH enzyme, per liter of culture. Thus, there remains a longstanding need in the art for improved methods for purifying OPH enzyme to provide the needed quantities for decontamination purposes. In addition, there is also a longstanding need for improved methods for the stable storage of OPH enzyme in order to allow for the stockpiling and transportation of the enzyme to locations requiring decontamination.
In order to solve these and other problems in the art, the present invention provides the following novel simplified purification processes, as well as a substantially enhanced yield of purified OP-hydrolyzing enzyme by the inventive processes, and other compositions and methods.
Accordingly, the invention provides a process for isolating organophosphorus hydrolase enzyme present in an aqueous solution of cell free bacterial proteins by contacting the aqueous solution of bacterial proteins with a strong cation exchange resin.
The strong cation exchange resin is then washed with a washing buffer to remove unbound proteins. Proteins that remain bound to the strong cation exchange resin are then eluted by washing the resin with an eluting buffer. The eluting buffer is prepared to include salt in a concentration that starts at about zero. The salt concentration of the eluting buffer is raised during the eluting process to about 0.5M, so that bound proteins are driven from the strong cation exchange resin in proportion to the salt concentration gradient.
The inventive process further includes detecting and collecting eluate, e.g., fractions, that include or contain a protein having organophosphorus hydrolase enzyme activity. In the process as exemplified herein, the protein having organophosphorus hydrolase enzyme activity is preferably detected in the eluate in collected fractions ranging in salt concentration from about 0.1 to about 0.2M. Preferably, the salt is NaCl, although any other suitable salt may be readily employed, e.g., any art-known salt that will displace organophosphorus hydrolase enzyme from the strong cation exchange resin without denaturing or deactivating the enzyme, and that is preferably non-toxic, including, simply by way of example, KCl, CaCl2, MgCl2, and the like.
Generally, for optimum storage and stability, the collected eluate is then precipitated with ammonium sulfate at a concentration effective to precipitate OPH, e.g., at about 45%, and then resuspended in a smaller volume of the same buffer, which is then dialyzed to further concentrate the OPH enzyme. The concentrated dialysate is then lyophilized to dryness. Surprisingly, it has been found that storage stability of the dried organophosphorus hydrolase enzyme prepared by the methods of the invention is greatly enhanced when the collected OPH enzyme is lyophilized in the presence of trehalose sugar. Preferably, the trehalose sugar is at a concentration of about 0.25M at the start of the lyophilization process.
The aqueous solution of cell free bacterial proteins is optionally pre-prepared or obtained from a commercial source, or more preferably, is prepared at the same location by cultivating recombinant bacteria, e.g., Escherichia coli (xe2x80x9cE. colixe2x80x9d), that express OPH enzyme in soluble form. The culture is grown to optimal density by art-standard methods, and in a preferred aspect of the invention, the opd gene is under the operable control of an inducible promoter, so that organophosphorus hydrolase enzyme production is induced after a suitable culture density is reached. As exemplified herein, the inducible promoter is a trc promoter. The cultivated recombinant bacteria are harvested, e.g., as a cell paste, and then lysed into a buffered aqueous solution, e.g., by passing the bacteria through a French Cell Press, freeze thawing and/or, ultrasonicating the bacteria Unlysed cells and cellular debris are separated, e.g., by filtration and/or centrifugation to provide an aqueous solution of cell free bacterial proteins.
The invention further provides for a composition that includes a soluble organophosphorus hydrolase enzyme bound to a strong cation exchange resin, such as, for example, Sepharose-SP(trademark) (Amersham Pharmacia Biotech, New Jersey).